Process for analysis of amino acids by liquid chromatography and color developing medium adapted therefor

ABSTRACT

A METHOD OF CHROMATOGRAPHY COMPRISING PASSING AMINO ACIDS TOGETHER WITH AN ELUTING SOLUTION THROUGH A SEPARATOR COLUMN PACKED WITH ION EXCHANGE RESINS, SEPARATING THE AMINO ACIDS INTO THEIR RESPECTIVE AMINO ACID COMPONENTS BY DIFFERENCES IN MIGRATION SPEEDS OF THE AMINO ACIDS IN THE COLUMN, ALLOWING THE SEPARATED AMINO ACIDS TO REACT WITH A COLOR-DEVELOPING SOLUTION CONTAINING NINHYDRINE AND ITS REDUCING AGENT BY HEATING THEREBY TO OBTAIN COLORED SUBSTANCES AND ANALYZING THE AMINO ACIDS HUES OF THE COLORED SUBSTANCES. THE COLOR-DEVELOPING SOLUTION CONTAINS A NAPHTHENIC DIAMINOTETRAACETIC ACID FOR MASKING HEAVY METALS.

Sept. 26, 1972 HISAYUKI SAGUSA ETAL 3,694,160

PROCESS FOR ANALYSIS OF AMINO ACIDS BY LIQUID CHROMATOGRAPHY AND COLORDEVELOPING MEDIUM ADAPTED THEREFOR Filed March 9, 1971 2 Sheets-Sheet 1FIG.

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PROCESS FOR ANALYSIS OF AMINO ACIDS BY LIQUID CHROMATOGRAPHY AND COLORDEVELOPING MEDIUM ADAPTED THEREFOR Filed March 9, 1971 2 Sheets-Sheet 2AMOUNT OF Cy 074 40050 (9/2) INVENTORS BY 0105 mam A/ZHLQL,

ATTORNEY$ United States Patent 3,694,160 PROCESS FOR ANALYSIS OF AMINOACIDS BY LIQUID CHROMATOGRAPHY AND COLOR DE- VELOPING MEDIUM ADAPTEDTHEREFOR Hisayuki Sagusa, Katsuta, and Seiji Takeuchi and YoshijiroArikawa, Hitachi, Japan, assignors to Hitachi, Ltd., Tokyo, Japan FiledMar. 9, 1971, Ser. No. 122,356 Claims priority, appligation Japan, Mar.11, 1970,

Int. Cl. G01u 31/08, 31/22 US. Cl. 23-230 R 7 Claims ABSTRACT OF THEDISCLOSURE This invention relates to an important in a method foranalyzing amino acids by liquid chromatography, and more particularly toan improvement in a liquid chromatography using ninhydrine as acolor-developing agent for detecting the amino acids.

In the liquid chromatography of amino acids, it is Well known that aminoacids are quantitatively determined by passing a sample of amino acidsthrough a column packed with ion exchange resins, separating the aminoacids into their respective components by utilizing differences in themigration speeds of the respective amino acid components in the column,allowing the separated amino acids to react with a color-developingagent and by measuring peaks of the developed colors of the reactedamino acids.

Heretofore, a classical method based on a manual operation by ananalyzing personnel called batch method has been available to effectcolor development of a sample of amino acids passed through the column,that is, an eluate solution, and effect a colorimetry of the eluatesolution, but recently a method for automatic colorimetry based on theuse of a through-flow type photometer capable of converting the detectedpeaks of colored substances into electric signals and recording the samehas been mainly used. For this automatic liquid chromatography, an ionexchange method such as Moor-Stein process, and a ligand method inventedby one of the present inventors of the present invention and disclosedin US. Pat. application Ser. No. 742,993 entitled Method of SeparatingMixture by Liquid Chromatography and filed May 14, 1968, now Patent No.3,630,681 are now used in practice.

According to the Moor-Stein method, a sample of amino acid mixture isseparated into the respective amino acid components by differences inthe migration speeds of the respective amino acid components when theamino acids pass through cation exchange resins if there are differencesin distribution coeflicient toward the cation exchange resins by thedifferences in degree of dissociation of the carboxyl groups of theamino acids. The thus separated amino acids are allowed to react with areducing agent, for example, a color-developing solution containingstannous chloride, SnCl and ninhydrine, and then 3,694,160 PatentedSept. 26, 1972 absorbances of colored substances resulting from thecolor-developing reaction are measured. For example, the maximumabsorption of Ruhemann purple is utilized for a-amino acid.

On the other hand, according to the ligand method, a metal salt-typecolumn, that is, ion exchange resin on which weakly basic metal ions,such as, Ni; Cu, Co++, Ca Hg++, Zn++, La+++, capable of forming acomplex salt with amino acids, etc., is adsorbed, is used, and the aminoacids are separated into their respective amino acid components bydifierences in the abilities of complex salt formation between variousamino acids and said metal ions when the amino acids are passed throughthe colmn. In the ligand method, color-developing agents other thanninhydrine can be used, but it is preferable, in view of thereactivities with about 20 kinds of amino acids resulting from thehydrolysis of protein, to use ninhydrine. Particularly in the automaticanalysis, it is very important in the efiiciency of analysis andperformance of apparatus that allamino acids can be analyzed only withone colordeveloping agent.

Ninhydrine is readily oxidized when exposed to air, and therefore it isnecessary to keep ninhydrine in a dark, cold place, while isolating itfrom air. Since an analytical system can be brought into a closed systemin the automatic analysis, there is no disadvantage in using theninhydrine.

A typical color-developing solution which has been employed has thefollowing composition:

Sodium acetate: 82 g./l.

Glacial acetic acid: 25 ml./l. (pH 5.5) Methyl cellosolve: 750 ml./l.Ninhydrine; 20 g./l. (0.1 M) SnCl -2H O: 0.4 g./l..(1.8X10" M) Sodiumacetate and glacial acetic acid are dissolved in 250 ml. of water.

Reaction of ninhydrine with an amino acid can be represented by thefollowing formula, where an intermediate reaction mechanism is omitted:

CO 00 U C=N-C U RCHO C02 Ruhemarm purple However, actual reaction is nota one-stage reaction as shown above, but its reaction mechanism can beassumed as follows: at first one molecule of the amino acid reacts withone molecule of ninhydrine to form diketohydrindanine,

through such intermdeiate products as Schitis bases, and further onemolecule of ninhydrine is combined with the diketohydrindamine to formRuhemann purple. Since the intermediate product is a reduciblesubstance, it will be consumed and the color-developing reaction will bedisturbed, if there is an oxidizable substance in the colordevelopingreaction system. The oxidizable substances which enter into the reactionsystem are, for example, dissolved oxygen, peroxide of methylcellosolveused as a solvent for ninhydrine and transition metals. It is easy toremove the dissolved oxygen or the peroxide from the reaction system,but it is impossible or very difiicult to completely remove a very smallamount of heavy metal ions such as Fe+++, Cu++, Mn++, Cr+++, which enterinto the reaction system at the preparation of an eluting solution orcolor-developing solution or from the separation system, etc.

Furthermore, the heavy metal ions form complex salts with the aminoacids or the intermediate product formed by reaction of the ninhydrine,i.e. hydrindantin, and Ruhemann purple, etc., and disturb the colordevelopment. Further, transition metals, among the heavy metals, oxidizea reducing agent as zinc powder, SnCl etc., which is added to thereaction system to form hydrindantin, and consume Zn and Sn++ as thereducing agent and form Zn++ and Sn++++. Therefore, the amount ofhydrindantin is reduced, and the efiiciency of color development islowered.

An object of the present invention is to provide an improved liquidchromatography, which can attain a high efllciency of color development.

Another object of the persent invention is to eliminate an influence ofoxidizable substances that disturb the color-developing reaction,particularly heavy metal ions in the liquid chromatography usingninhydrine as a colordeveloping agent.

Still another object of the present invention is to provide a usefulmeans for masking the heavy metal ions in an automatic analysis of aminoacids by liquid chromatography.

Further object of the present invention is to provide a useful means formasking the heavy metal ions in a liquid chromatography based on theligand method, using a metal salt-type column.

Still further object of the present invention is to pro vide a usefulninhydrine color-developing solution in a liquid chromatography by theligand method.

To attain these and other objects which will be seen from the followingdetailed description, the present invention provides an improved methodof adding naphthenic diaminotetraacetic acid to a color-developingsystem to mask heavy metals that disturb reaction of amino acids withninhydrine in a method for analyzing amino acid by liquidchromatography, which comprises separating various amino acids intotheir respective components by differences in migration speeds of theamino acids in a column packed with ion exchange resins, allowing thethus separated amino acids to react with a ninhydrine color-developingsolution thereby to form colored substances, and efiecting colorimetryof the colored substances.

The present invention is based on a novel concept of masking heavymetals that disturb the color-developing reaction between amino acidsand ninhydrine, by a suitable complex-forming agent. However, therestrictions required for the complex-forming agent are too various andstrict as a practical matter to make a proper selection of thecomplex-forming agent with ease. For example, it is, of course,necessary to select a complex-forming agent that gives no unfavorableinfluence upon the ninhydrine in the color-developing solution and hasan ability good enough to form complexes with heavy metal ions, but inthe liquid chromatography based on the ligand method, an organicsolvent, for example, 75 vol. percent methylcellosolve, is used tosufficiently dissolve hydrindantin. That is, the compex-forming agentmust be sufficiently dissolved in such an organic solvent. In thisrespect, ethylenediaminetetraacetic acid, a well-known complex-formingagent, cannot be used, because it cannot be dissolved sufficiently inthe organic solvent. Further, glycoletherdiaminetetraacetic acid ordiethylenetriaminepentaacetic acid is likewise insoluble in the solvent.Amine complex-forming agents such as ethanol amine are positive to theninhydrine, and both amine complex-forming agent and ninhydrine reactwith each other. Phenanthroline or tripyridyltriazine forms coloredcompounds with most of metal ions and thus cannot be used.

In brief, the conditions required for the complex-forming agent ormasking agent are as follows:

(1) It must have a good ability to form complex compounds with the heavymetal ions.

(2) It must be negative to the color development of ninhydrine.

(3) The resulting complex compounds with the heavy metal ions must becolorless.

(4) It must be sufliciently soluble in such an organic solvent asmethyleellosolve.

The present inventors have found that naphthenic diaminetetraaceticacids are suitable as the complex-forming agent that can meet theseconditions. The naphthenic diaminetetraacetic acids includecyclohexanediaminetetraacetic acid (which will be hereinafter referredto as CyDTA), cyclohexanoldiaminetetraacetic acid,methylcyclohexanediaminetetraacetic acid, cyclopentanediaminetetraaceticacid, etc.

In the ion exchange method, the naphthenic diaminetetraacetic acids canbe added to the ninhydrine colordeveloping solution and/or elutingsolution, but in the ligand method, it is practically not suitable toadd this complex-forming agent to the eluting solution, because, whenthe complex-forming agent is passed through the metal salt-type column,it forms complex salts with the metals adsorbed on the ion exchangeresin, and the metal ions that are to form complex salts with the aminoacids are consumed. In other words, an equilibrium of the column isbrought to disorder, when the naphthenic diaminetetraacetic acid isadded to the eluting solution. Therefore, such a procedure must beavoided.

In the ligand method, the color-developing reaction is sometimesdisturbed, depending upon the kind of metal ions to be adsorbed on theion exchange resins. That is to say, metal ions form chelate compoundswith amino acids in the column, and the resulting chelate compoundsreact with hydrindantin to form reaction products of hydrindantin withamino acids, that is, Ruhemann purple. However, the liberated metal ionssometimes disturb the formation reaction of Ruhe'mann purple. In thatcase, the naphthenic diaminotetraacetic acid is added thereto in anamount enough to mask the metal ions. In other words, when a metalsalt-type column on which metals disturbing the color development, forexample, Cu+ are added, is used, the naphthenic diaminetetraacetic acidis added thereto in an amount necessary for masking all the heavy metalions to be joined as impurities and the metal ions to be eluted from thecolumn. However, Zinc ion gives no influence upon hydrindantin or thereducing agent, and thus in that case, the naphthenic diaminetetraaceticacid is added thereto only in an amount enough to mask the heavy metalions and the metal ions used as the reducing agent.

Embodiments of the present invention will be explained, by reference tothe accompanying drawings:

FIG. 1 is a schematic diagram of an automatic analyzing apparatus usedin the present invention;

FIG. 2 is a schematic view of a through-flow type detector; and

FIG. 3 is a graph showing a relation between the color development ratioand the amount of cyclohexanediaminetetraacetic acid added according tothe present invention.

In FIG. 1, strongly acidic resins of sulfonic acid formed within acolumn 1 are brought into an equilibrium with an eluting solutioncontaining 10= M./l. of Zn++. The eluting solution stored in a vessel 3is fed to the columnby a pump 2. The eluting solution contains 10 M./l.of Zn++ and also contains an acetic acid-sodium acetate buffer solution(pH 5.2) adjusted so that the Na+ concentration may be 0.9 M./l. Areactor 8, a through-flow type photometer 4 and a recorder 5 areconnected in succession to the etfiuent side of the column 1. In avessel 7, a ninhydrine color-developing solution is stored, and is addedto a passage for a sample solution leaving the column as an eluateefiiuent by means of a pump 6. The composition of a color-developingsolution is 82 g./l. of sodium acetate, 25 ml./l.) of glacial aceticacid (pH 5.5), 750 ml./l. (0.1 M./l.) of methylcellosolve, 20 g./l. (0.1M./l.) of ninhydrine, 0.4 g./l. (1.8 10- M./l.) of SnCl; and CyDTA, thebalance being Water. The feeding rates of the pumps 2 and 6 are 90ml./hour and 45 ml./ hour, respectively.

The eluting solution is allowed to pass through the column 1 by means ofthe pump 2 to bring the system of Zn++-adsorbed ion exchange resins intoan equilibrium. Then, an amino acid sample is added to the top of thecolumn 1. The amino acid sample is separated into the respective aminoacid components by dilferences in coordinate-bonding forces between therespective amino acid components and Zn++ adsorbed on the ion exchangeresins, that is, by difference in the abilities to form complex salts,and further by the resulting differences in the migration speeds withinthe column 1, and flows as an eluate eflluent from the bottom, that is,the effluent side of the column 1. The color-developing solution isadded to the eluate efiluent by means of the pump 6, and then theresulting mixture is led to the reactor 8 and heated, whereby ninhydrineis allowed to react with the amino acids (more exactly hydrindantinformed by the reduction of ninhydrine by the reducing agent SnCl in thereactor reacts with amino acids) and form colored substances. Thesolution containing the colored substances is led to the through-flowtype photometer 4, where absorbances of the colored substancescorresponding to the respective amino acid components are continuouslydetected. The thus detected absorbances are converted to electricsignals, which are recorded in a recorder 5.

As shown in FIG. 2, the through-flow photometer is provided with a flowcell 12, through which the solution containing the colored substances isallowed to pass. When light is projected onto the fiow cell from a lightsource 11, the light is absorbed by the colored substances, and thetransmitted light is detected by a light-detecting means 14 such as aphotocell, etc. The light signal is converted to electric signals, whichare amplified by an amplifier 16 and led to the recorder 5 as theelectric signals. In the automatic analytical method, a means forcontinuously detecting and analyzing light signals corresponding 'to therespective amino acid components by means of such a flow cell isindispensable.

In FIG. 3, color development ratio of a ninhydrine color-developingsolution is shown when an amount of CyDTA, which is added as thecomplex-forming agent, is changed. The compositions of the elutingsolution and the color-developing solution and the feeding rates are thesame as above. The amount of amino acid sample is 0.4 ,ug. The reactionin the reactor is carried out at 115 C. for 3 minutes. The heatingmedium for the reactor is glycerine. In FIG. 3, the color developmentratio is represented by an area of a peak of the recorded absorbance, inother words, by a product, /2 h. W that is, a half height of peak, /2h., multiplied by a peak width, W at said h'alf height of the peak (Ah.)

In FIG. 3, a curve I shows the color development ratio for glycerine, acurve II tryrosine, a curve III arginine, and a curve IV ammonia. Itshould be noted that ammonia is included in the amino acid as aresemblance to the amino acid. It is seen from the graph that the colordevelopment ratio is increased by an increase in the amount of CyDTAadded, but the color development ratio becomes constant, when the amountof CyDTA exceeds 0.6 g./l. (1.8)( M./l.). This means that all the heavymetal ions are masked. Even if CyDTA is added thereto in excess, itgives no disturbance to the color-development reaction. That is, CyDTAis easy to use, and therefore it is preferable to use more than 0.6g./l. of CyDTA in order to completely mask the heavy metals. If thecolor development ratio is assumed when no CyDTA is added, the colordevelopment ratios, when 0.6 g./l. or more of CyDTA is added thereto,are 126% for glycine, 138% for tryosene, 140% for arginine and 166% forammonia. That is, the color development ratios are considerablyincreased by adding @DTA thereto.

In the foregoing example, an embodiment of using SnCl as a reducingagent is exemplified. However, it has been found that metal zinc powderslikewise can be used. The metal zinc powders have a higher solubility inthe color-developing solution that SnCl and therefore have such anadvantage that the metal zinc powders never produce any precipitate.

What is claimed is:

1. In a method for analyzing amino acids by liquid chromatography, whichcomprises passing amino acids together with an eluting solution througha separator column packed with ion exchange resins, separating the aminoacids into their respective amino acid components by differences inmigration speeds of the amino acids in the column, allowing theseparated amino acids to react with a color-developing solutioncontaining ninhydrine and its reducing agent by heating thereby toobtain colored substances and analyzing the amino acids by hues of thecolored substances, an improvement comprising adding a naphthenicdiaminotetraacetic acid to the color-developing solution as an agent formasking heavy metal ions.

2. In a method for automatically analyzing amino acids by liquidchromatography, which comprises passing amino acids together with aneluting solution through a separator column packed with ion exchangeresins, separating the amino acids into their respective amino acidcomponents by diiferences in migration speeds of the amino acids in thecolumn, allowing the separated amino acids to react with acolor-developing solution containing ninhydrine and its reducing agentby heating thereby to obtain colored substances, leading the resultingsolution containing said colored substances continuously to a means fordetecting absorbances and transmitting detected signals from the meansfor detecting absorbances to a recording means, an improvementcomprising adding a proper amount of a naphthenic diaminotetraaceticacid as an agent for masking metal ions capable of disturbing theformation of said colored substances, to an analytical system at leastat a stage prior to the formation reaction of said colored substances.

3. In a method for automatically analyzing amino acids by liquidchromatography, which comprises passing an eluting solution containing abuffer agent and the same metal ions as those adsorbed on ion exchangeresins in a separator column thereby to bring the column into anequilibrium, adding an amino acid sample to the separator column therebyto separate amino acids into the respective amino acid components bymigration speeds due to differences in abilities to form complex saltsof the respective amino acids with the metal ions, mixing an effiuenteluate solution leaving the column with a colordeveloping solutioncontaining ninhydrine and its reducing agent, heating the resultingmixture solution in a reactor to carry out a color-developing reaction,continuously passing the resulting colored substance Ruhemann purple toa flow cell, where light is projected onto the solution and lightabsorption by the colored substance is effected, detecting a quantity oflight, converting the detected quantity of light to electric signals andrecording the electric signals, reaction system within the analyticalsystem being isolated from surrounding atmosphere, an improvementcomprising adding an eifective amount of a naphthenic diaminotetraaceticacid to the color-developing solution as an agent for masking metal ionscapable of disturbing the color development reaction.

4. An improvement according to claim 3, wherein the metal ions adsorbedon the ion exchange resins in the column are non-oxidizable metal ionsincapable of disturbing the color development reaction, and the amountof the naphthenic diaminotetraacetic acid to be added is at least about1.8 10 moles/l.

5. An improvement according to claim 3, wherein the amount of thenaphthenic diaminotetraacetic acid is an amount necessary for formingcomplex compounds with all heavy metal ions entered in the system asimpurities and metal ions adsorbed on the ion exchange resins in thecolumn when the latter metal ions are oxidizable toward a reductionproduct of ninhydrine, hydrindantin.

6. A color-developing solution for analyzing amino acids by liquidchromatography, which comprises ninhydrine, a buffer agent, a reducingagent for ninhydrine, an

5 aminotetraacetic acid is contained.

References Cited UNITED STATES PATENTS 9/1968 Skeggs et a1. 23-230 RMORRIS O. WOLK, Primary Examiner R. M. REESE, Assistant Examiner

